metallothionein and Hypoxia

Metal responsive transcription factor 1 (MTF-1) is a zinc dependent transcription factor which is involved in the regulation of intracellular signaling pathways. MTF-1 regulates the expression of two streams of genes functioning in metal homeostasis and anti-oxidative response. MTF-1 acts in the process of binding of toxic metal ions in the cell, due to the activation of the expression of metallothioneins (MTs). Additionally, MTF-1 regulates transcription of genes involved in the sequestration of zinc and its intracellular transport. Disruption of zinc and MT homeostasis has an indispensable influence on the development of several pathological states. Moreover, by increasing MT activity, MTF-1 can effectively protect cells from oxidative and hypoxic stresses. The mechanism of MTF-1 action in cells includes the regulation of the proper immune response through activation/repression of anti- and pro-inflammatory cytokines. MTF-1 function in immune response is related to nuclear factor-κB (NF-κB) activity. Synthesis of insulin is also related to the activity of this transcription factor and zinc balance. Insulin transport also depends on zinc. In pancreatic β-cells, several types of the zinc transporters are found. Zinc transporters coordinated action is crucial for the synthesis and secretion of insulin. Disturbances in the regulation of signaling pathways connected with MTF-1 function can entail further alterations in zinc intracellular status and this growing imbalance can promote the pathophysiology of degenerative disorders.

Topics: Animals; Cytokines; DNA-Binding Proteins; Drosophila; Homeostasis; Humans; Hypoxia; Inflammation; Insulin; Insulin-Secreting Cells; Metallothionein; Mice; NF-kappa B; Nuclear Localization Signals; Oxidative Stress; Promoter Regions, Genetic; Protein Structure, Tertiary; Signal Transduction; Transcription Factor MTF-1; Transcription Factors; Zinc

The metal-responsive transcription factor-1 (MTF-1, also termed MRE-binding transcription factor-1 or metal regulatory transcription factor-1) is a pluripotent transcriptional regulator involved in cellular adaptation to various stress conditions, primarily exposure to heavy metals but also to hypoxia or oxidative stress. MTF-1 is evolutionarily conserved from insects to humans and is the main activator of metallothionein genes, which encode small cysteine-rich proteins that can scavenge toxic heavy metals and free radicals. MTF-1 has been suggested to act as an intracellular metal sensor but evidence for direct metal sensing was scarce. Here we review recent advances in our understanding of MTF-1 regulation with a focus on the mechanism underlying heavy metal responsiveness and transcriptional activation mediated by mammalian or Drosophila MTF-1. This article is part of a Special Issue entitled: Cell Biology of Metals.

Topics: Amino Acid Sequence; Animals; Conserved Sequence; DNA-Binding Proteins; Homeostasis; Humans; Hypoxia; Metallothionein; Metals, Heavy; Molecular Sequence Data; Oxidative Stress; Protein Structure, Tertiary; Signal Transduction; Transcription Factor MTF-1; Transcription Factors; Transcriptional Activation

Superoxide formation in pulmonary tissue is modulated by cytokines, PO2, shear force, and disease states, and can be stimulated by drugs. Superoxide has diverse actions on pulmonary cells, including smooth muscle contraction, interaction with redox enzymes, cell proliferation, and gene transcription. In the lungs, there is an impressive array of specific defence mechanisms that destroy superoxide, especially superoxide dismutase (SOD) and metallothionein. Superoxide formation is increased in hyperoxia (e.g., oxygen therapy); however, superoxide-forming enzymes also can be up-regulated in hypoxia. Superoxide has been implicated in acute respiratory distress syndrome, lung ischaemia-reperfusion injury, and lung transplantation. Novel approaches to therapy have been explored, including SOD gene therapy and SOD targeting to the lung. In the future, new drugs interacting with superoxide may provide significant advances in the treatment of lung diseases.

Topics: Animals; Cell Division; Cell Membrane Permeability; Genetic Therapy; Humans; Hyperoxia; Hypoxia; Lung Diseases; Metallothionein; Pulmonary Artery; Reperfusion Injury; Superoxide Dismutase; Superoxides; Transcription, Genetic

The increased blood stasis and venous volume pressure causing tissue hypoxia are observed in both varicocele and varicose veins. Metallothionein (MT), a metal-binding protein, protects against cell apoptosis under hypoxic stress. It also plays an important role in collateral flow recovery and angiogenesis. We studied the distribution of hypoxia-inducible factor-1α (HIF-1α) and MT in varicocele and varicose veins.. The study specimens consisted of 1 cm venous segments that were obtained from 12 male patients during vascular stripping surgery for varicose veins and 1 cm of internal spermatic vein (ISV) obtained from 12 patients during left varicocele repair. The control samples of 1 cm ISV were obtained from 10 male patients who underwent left inguinal herniorrhaphy. All vascular specimens were analysed for HIF-1α and MT expression by immunoblotting, immunohistochemical (IHC) staining and confocal microscopy. Data were analysed using one-way analysis of variance with Tukey's comparison test.. In both venous diseases, the increased expression of HIF-1α and MT compared with the control group (P < 0.05) and most of the proteins distributed over smooth muscle layers were detected by IHC staining; HIF-1α and MT in the muscle layer with co-localization, and MT overexpression especially located in the endothelium of both venous diseases under confocal microscopy.. Our results revealed the higher expression of HIF-1α and MT in varicocele and varicose veins than in the control group; MT overexpression in the muscle layer of both diseased vessels and especially located in the endothelium under confocal microscopy. MT has the function to protect vascular cells from apoptosis under hypoxia. Thus, this MT function may cause a decreased vascular cell apoptosis and then contribute to the dilated and thickened walls of varicocele and varicose veins.

Topics: Adult; Apoptosis; Endothelium, Vascular; Female; Gene Expression Regulation; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Metallothionein; Middle Aged; Muscle, Smooth, Vascular; Stress, Physiological; Varicocele; Varicose Veins

The reperfusion of coronary artery blood supply is often accompanied by myocardial hypoxia/reperfusion (H/R) injury, and induced cardiomyocytes apoptosis. The activation of p38 can induce apoptosis, thereby aggravating the myocardial H/R injury. Metallothionein-2A (MT2A) has the functions of anti-apoptosis and protective effect through p38. However, it is not clear that MT2A may protect cardiomyocytes from H/R injury through p38 signaling pathway. Here, we constructed an H/R model for H9c2 cardiomyocytes to explore the protective effect of MT2A on cardiomyocytes apoptosis during the process of H/R through p38 signal pathway. The results revealed that both endogenously overexpressed MT2A and exogenously added MT2A can inhibit the active expression of p-p38 and cleaved caspase-3 under H/R. Based on our results, H/R induced cardiomyocytes apoptosis and activation of p38. And, MT2A can inhibit the active expression of caspase-3 and p38. We found that MT2A can protect cardiomyocytes apoptosis from H/R injury through p38 signaling pathway.

Topics: Caspase 3; Cell Hypoxia; Humans; Hypoxia; Metallothionein; Myocardial Reperfusion Injury; Myocytes, Cardiac

Obstructive sleep apnea (OSA) is an independent risk factor for cardiovascular disease. While intermittent hypoxia (IH) and catecholamine release play an important role in this increased risk, the mechanisms are incompletely understood. We have recently reported that IH causes endothelial cell (EC) activation, an early phenomenon in the development of cardiovascular disease, via IH-induced catecholamine release. Here, we investigated the effects of IH and epinephrine on gene expression in human aortic ECs using RNA-sequencing. We found a significant overlap between IH and epinephrine-induced differentially expressed genes (DEGs) including enrichment in leukocyte migration, cytokine-cytokine receptor interaction, cell adhesion and angiogenesis. Epinephrine caused higher number of DEGs compared to IH. Interestingly, IH when combined with epinephrine had an inhibitory effect on epinephrine-induced gene expression. Combination of IH and epinephrine induced MT1G (Metallothionein 1G), which has been shown to be highly expressed in ECs from parts of aorta (i.e., aortic arch) where atherosclerosis is more likely to occur. In conclusion, epinephrine has a greater effect than IH on EC gene expression in terms of number of genes and their expression level. IH inhibited the epinephrine-induced transcriptional response. Further investigation of the interaction between IH and epinephrine is needed to better understand how OSA causes cardiovascular disease.

Topics: Aorta; Cardiovascular Diseases; Cytokines; Endothelial Cells; Epinephrine; Humans; Hypoxia; Metallothionein; Receptors, Cytokine; RNA; Sleep Apnea, Obstructive

The white shrimp Litopenaeus vannamei is exposed to hypoxic conditions in natural habitats and in shrimp farms. Hypoxia can retard growth, development and affect survival in shrimp. The hypoxia-inducible factor 1 (HIF-1) regulates many genes involved in glucose metabolism, antioxidant proteins, including metallothionein (MT) and apoptosis. In previous studies we found that the L. vannamei MT gene expression changed during hypoxia, and MT silencing altered cell apoptosis; in this study we investigated whether the silencing of HIF-1 affected MT expression and apoptosis. Double-stranded RNA (dsRNA) was used to silence HIF-1α and HIF-1β under normoxia, hypoxia, and hypoxia plus reoxygenation. Expression of HIF-1α, HIF-1β and MT, and apoptosis in hemocytes or caspase-3 expression in gills, were measured at 0, 3, 24 and 48 h of hypoxia and hypoxia followed by 1 h of reoxygenation. The results showed that hemocytes HIF-1α expression was induced during hypoxia and reoxygenation at 3 h, while HIF-1β decreased at 24 and 48 h. In normoxia, HIF-1 silencing in hemocytes increased apoptosis at 3 h and decreased at 48 h; while in gills, caspase-3 increased at 3, 24 and 48 h. In hypoxia, HIF-1 silencing decreased apoptosis in hemocytes at 3 h, but caspase-3 increased in gills. During reoxygenation, apoptosis in hemocytes and caspase-3 in gills increased. During normoxia in hemocytes, silencing of HIF-1 decreased MT expression, but in gills, MT increased. During hypoxia and reoxygenation, silencing induced MT in hemocytes and gills. These results indicate HIF-1 differential participation in MT expression regulation and apoptosis during different oxygen conditions.

Topics: Animals; Apoptosis; Aryl Hydrocarbon Receptor Nuclear Translocator; Fish Proteins; Gene Expression Regulation; Gills; Hemocytes; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Metallothionein; Oxygen; Penaeidae; Reactive Oxygen Species

Intermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), is prevalent in older adults and associated with inflammation. We previously showed that IH induces renal fibrosis and cardiomyopathy and hypothesized that lung inflammatory changes may underlie deficits in pulmonary function in OSA.. Pulmonary inflammatory and oxidative markers were assessed in metallothionein KO (MT-KO) mice and WT 129S1 controls exposed to IH or to normoxia for 8 weeks.. MT expression increased at 3 days in WT, falling back at 1 week. Pro-fibrotic markers CTGF and PAI-1 were unchanged in WT, but increased at 3 or 8 weeks, with enhanced Sirius Red staining at 8 weeks, in IH-exposed MT-KO. Cellular infiltration, TNF-α and IL-6 increased earlier in IH-exposed MT-KO than in WT. Oxidative markers, 3-nitrotyrosine and 4-hydroxynonenal increased in both but persisted in MT-KO. Antioxidant Nrf2, HO-1 and NQO1, increased at 3 days in WT mice and at 8 weeks IH in MT-KO. While early Nrf2 induction required MT, its later increase at 8 weeks in MT-KO was independent from MT.. We conclude that early MT and antioxidant gene response protects from fibrotic changes in long-term IH-exposed mouse lung. Without this response, pulmonary fibrosis may develop with longer IH exposure.

Topics: Animals; Hypoxia; Interleukin-6; Lung; Lung Injury; Metallothionein; Mice, Knockout; Oxidative Stress; Tumor Necrosis Factor-alpha

The choroid plexus (CP) is a key regulator of the central nervous system (CNS) homeostasis through its secretory, immunological and barrier properties. Accumulating evidence suggests that the CP plays a pivotal role in the pathogenesis of multiple sclerosis (MS), but the underlying mechanisms remain largely elusive. To get a comprehensive view on the role of the CP in MS, we studied transcriptomic alterations of the human CP in progressive MS and non-neurological disease controls using RNA sequencing. We identified 17 genes with significantly higher expression in progressive MS patients relative to that in controls. Among them is the newly described long non-coding RNA HIF1A-AS3. Next to that, we uncovered disease-affected pathways related to hypoxia, secretion and neuroprotection, while only subtle immunological and no barrier alterations were observed. In an ex vivo CP explant model, a subset of the upregulated genes responded in a similar way to hypoxic conditions. Our results suggest a deregulation of the Hypoxia-Inducible Factor (HIF)-1 pathway in progressive MS CP. Importantly, cerebrospinal fluid levels of the hypoxia-responsive secreted peptide PAI-1 were higher in MS patients with high disability relative to those with low disability. These findings provide for the first time a complete overview of the CP transcriptome in health and disease, and suggest that the CP environment becomes hypoxic in progressive MS patients, highlighting the altered secretory and neuroprotective properties of the CP under neuropathological conditions. Together, these findings provide novel insights to target the CP and promote the secretion of neuroprotective factors into the CNS of progressive MS patients.

Topics: Adrenomedullin; Adult; Aged; Case-Control Studies; Choroid Plexus; Female; Gene Expression Profiling; Gene Ontology; Glycoproteins; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Intercellular Signaling Peptides and Proteins; Lateral Ventricles; Male; Metallothionein; Middle Aged; Multiple Sclerosis, Chronic Progressive; Multiple Sclerosis, Relapsing-Remitting; Neuroprotection; Neurosecretion; Plasminogen Activator Inhibitor 1; RNA-Seq; RNA, Antisense; RNA, Long Noncoding

Nuclear factor-E2-related factor 2 (Nrf2) and metallothionein have each been reported to protect against chronic intermittent hypoxia- (IH-) induced cardiomyopathy. Sulforaphane-rich broccoli sprout extract (BSE) and zinc can effectively induce Nrf2 and metallothionein, respectively, to protect against IH-induced cardiomyopathy via antioxidative stress. However, whether the cardiac protective effects of the combination of BSE and zinc can be synergistic or the same has not been evaluated. In this study, we treated 8-week-old C57BL/6J mice with BSE and/or zinc during exposure to IH for 8 weeks. Cardiac dysfunction, as determined by echocardiography, and pathological remodeling and abnormalities, including cardiac fibrosis, inflammation, and oxidative damage, examined by histopathology and western blotting, were clearly observed in IH mice but were not significant in IH mice treated with either BSE, zinc, or zinc/BSE. Furthermore, the effects of the combined treatment with BSE and zinc were always greater than those of single treatments. Nrf2 function and metallothionein expression in the heart increased to a greater extent using the combination of BSE and zinc than using BSE or zinc alone. These findings for the first time indicate that the dual activation of Nrf2 and metallothionein by combined treatment with BSE and zinc may be more effective than monotherapy at preventing the development of IH-induced cardiomyopathy.

Topics: Animals; Antioxidants; Brassica; Cardiomyopathies; Drug Therapy, Combination; Humans; Hypoxia; Metallothionein; Mice; Mice, Inbred C57BL; NF-E2-Related Factor 2; Oxidative Stress; Plant Extracts; Sleep Apnea, Obstructive; Zinc

We reported previously that nuclear factor erythroid 2-related factor 2 (Nrf2) and metallothionein (MT) play critical roles in preventing intermittent hypoxia (IH)-induced cardiomyopathy. In addition, positive feedback regulation between Nrf2 and MT is required for the efficient compensative responses of the heart to IH. As an activator of Nrf2, sulforaphane (SFN) has attracted attention as a potential protective agent against cardiovascular disease. Here, we investigated whether SFN can up-regulate cardiac Nrf2 expression and function, as well as MT expression, to prevent IH-induced cardiomyopathy, and if so, whether Nrf2 and MT are indispensable for this preventive effect. Nrf2-knock-out (Nrf2-KO) or MT-KO mice and their wild-type (WT) equivalents were exposed to IH for 4 weeks with or without SFN treatment. SFN almost completely prevented IH-induced cardiomyopathy in WT mice, and this preventive effect was abolished in Nrf2-KO mice but retained in MT-KO mice. In IH-exposed WT mice, SFN induced significant increases in the expression levels of Nrf2 and its downstream antioxidant target genes, as well as those of MT, but these effects were not seen in IH-exposed Nrf2-KO mice. By contrast, KO of MT did not affect the ability of SFN to up-regulate the expression of Nrf2 and its downstream antioxidant targets. These results suggest that SFN-induced MT expression is Nrf2-dependent, and SFN prevents IH-induced cardiomyopathy in a Nrf2-dependent manner, for which MT is dispensable. This study provides important information that is relevant to the potential use of SFN to prevent IH-induced cardiomyopathy.

Topics: Animals; Cardiomyopathies; Cardiotonic Agents; Gene Expression Regulation; Hypoxia; Isothiocyanates; Male; Metallothionein; Mice; Mice, Knockout; NF-E2-Related Factor 2; Sulfoxides

The mechanism for intermittent hypoxia (IH)-induced cardiomyopathy remains obscure. We reported the prevention of acute and chronic IH-induced cardiac damage by selective cardiac overexpression of metallothionein (MT). Herein we defined that MT-mediated protection from IH-cardiomyopathy is via activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a critical redox-balance controller in the body. For this, mice were exposed to IH for 3 days (acute) or 4 or 8 weeks (chronic). Cardiac Nrf2 and MT expression in response to IH were significantly increased acutely yet decreased chronically. Interestingly, cardiac overexpression (Nrf2-TG) or global deletion of the Nrf2 gene (Nrf2-KO) made mice highly resistant or highly susceptible, respectively, to IH-induced cardiomyopathy and MT expression. Mechanistically, 4-week IH exposure significantly decreased cardiac Nrf2 binding to the MT gene promoter, and thus, depressed both MT transcription and translation in WT mice but not Nrf2-TG mice. Likewise, cardiac MT overexpression prevented chronic IH-induced cardiomyopathy and down-regulation of Nrf2 likely via activation of a PI3K/Akt/GSK-3β/Fyn-dependent signaling pathway. These results reveal an integrated relationship between cardiac Nrf2 and MT expression in response to IH -- acute compensatory up-regulation followed by chronic down-regulation and cardiomyopathy. Cardiac overexpression of either Nrf2 or MT offered cardioprotection from IH via complicated PI3K/Akt/GSK3B/Fyn signaling. Potential therapeutics may target either Nrf2 or MT to prevent chronic IH-induced cardiomyopathy.

Topics: Animals; Cardiomyopathies; Gene Expression Regulation; Glycogen Synthase Kinase 3 beta; Hypoxia; Male; Metallothionein; Mice; Mice, Transgenic; NF-E2-Related Factor 2; Oxidation-Reduction; Oxidative Stress; Phosphatidylinositol 3-Kinases; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-fyn; Signal Transduction

As a main clinical feature of obstructive sleep apnea (OSA), intermittent hypoxia (IH) induces oxidative stress, leading to damage to a variety of organs, including kidney. Metallothionein (MT) is a potent antioxidant that protects kidney against oxidative damage. Our previous studies demonstrated that MT prevented IH-induced cardiomyopathy in mice. However, the role of MT in protecting against IH-induced renal injury is unknown. Therefore, MT knockout (MT KO) mice and wild type (WT) control mice (129S) were culled for exposure to intermittent air as control or IH for a time course of 3 days, 1 week, 3 weeks and 8 weeks. MT KO mice developed higher urinary albumin to creatinine ratio (UACR) after exposure to IH for 8 weeks. Compared with either MT KO control or WT IH mice, MT deletion significantly aggravated IH-induced renal oxidative damage and inflammation at all four time points, along with significant acceleration of renal fibrosis after exposure to IH for 3 weeks and 8 weeks. Antioxidants including MT, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), heme oxygenase 1 (HO1) and NAD (P) H dehydrogenase [quinone] 1 (NQO1) were increased in response to short-term IH (3 days, 1 week and 3 weeks) but decreased after long-term IH (8 weeks) in WT mice. Interestingly, Nrf2, HO1 and NQO1 were significantly attenuated under IH conditions in the absence of MT, which were in parallel with the inactivation of protein kinase B (Akt) and extracellular signal-regulated kinase (ERK). These findings demonstrated that MT played a key role in preventing IH-induced renal injury possibly via preserving Nrf2 signaling pathway.

Topics: Acute Kidney Injury; Albuminuria; Animals; Antioxidants; Fibrosis; Gene Deletion; Hypoxia; Kidney Function Tests; Lipid Peroxidation; Metallothionein; Mice; Mice, Knockout; Nephritis

The pacu (Piaractus mesopotamicus) is a hypoxia-tolerant neotropical fish species. There is little or no information in this species regarding biochemical adaptations to waters with different oxygen concentrations, such as the production of reactive oxygen species and antioxidant scavengers, which might be of interest in the study of antioxidant defense mechanisms. Metallothioneins (MT) have been widely applied as biomarkers for metal exposure in fish liver, and, recently, in bile. These metalloproteins, however, have also been reported as free radical scavengers, although studies in this regard are scarce in fish. In this context, normoxic and hypoxic controlled experiments were conducted with pacu specimens and MT levels were quantified in both liver and bile. Reduced glutathione (GSH) indicative of oxidative stress, and thiobarbituric acid reactive substances (TBARS), indicative of lipid peroxidation, were also determined in liver. The results demonstrate that hypoxic fish present significantly lower metallothionein levels in liver and bile and lower reduced glutathione levels in liver, whereas lipid peroxidation was not significantly different between hypoxic and normoxic fish. The results of the present study seem to suggest that metallothioneins may actively participate in redox regulation in hypoxic fish in both bile and liver. MT levels in these organs may be temporarily suppressed, supporting the notion that down-regulation of oxidant scavengers during the oxidative burst is important in defense signaling in these adapted organisms.

Topics: Animals; Bile; Characiformes; Glutathione; Hypoxia; Lipid Peroxidation; Liver; Metallothionein; Oxidation-Reduction; Oxidative Stress; Spectrophotometry; Thiobarbituric Acid Reactive Substances

Obstructive sleep apnea (OSA) causes chronic intermittent hypoxia (IH) to induce cardiovascular disease, which may be related to oxidative damage. Metallothionein (MT) has been extensively proved to be an endogenous and highly inducible antioxidant protein expressed in the heart. Therefore, we tested the hypotheses that oxidative stress plays a critical role in OSA induced cardiac damage and MT protects the heart from OSA-induced cardiomyopathy. To mimic hypoxia/reoxygenation events that occur in adult OSA patients, mice were exposed to IH for 3 days to 8 weeks. The IH paradigm consisted of alternating cycles of 20.9% O₂/8% O₂ F(I)O₂ (30 episodes per hour) with 20s at the nadir F(I)O₂ for 12 h a day during daylight. IH significantly increased the ratio of heart weight to tibia length at 4 weeks with a decrease in cardiac function from 4 to 8 weeks. Cardiac oxidative damage and fibrosis were observed after 4 and 8 weeks of IH exposures. Endogenous MT expression was up-regulated in response to 3-day IH, but significantly decreased at 4 and 8 weeks of IH. In support of MT as a major compensatory component, mice with cardiac overexpression of MT gene and mice with global MT gene deletion were completely resistant, and highly sensitive, respectively, to chronic IH induced cardiac effects. These findings suggest that chronic IH induces cardiomyopathy characterized by oxidative stress-mediated cardiac damage and the antioxidant MT protects the heart from such pathological and functional changes.

Topics: Animals; Cardiomyopathies; Hypoxia; Male; Metallothionein; Mice; Myocardium; Oxidation-Reduction; Oxidative Stress; Sleep Apnea, Obstructive

Endoplasmic reticulum (ER) stress, an adaptive response normally, causes apoptotic cell death under pathological conditions. Cardiac ER stress and associated cell death involve in the inflammatory responses that often cause cardiac remodeling and dysfunction. Here we examined whether chronic intermittent hypoxia (IH) induces cardiac ER stress and associated cell death along with inflammatory response and if so, whether these effects can be affected by transgenic overexpression or deletion of metallothionein gene (MT-TG or MT-KO). IH exposures for 3 days to 4 weeks significantly increased cardiac ER stress and apoptosis, shown by the increased expression of GRP78, ATF6 and CHOP, the activation of caspase-12 and capase-3, and the decreased Bcl2/Bax expression ratio, predominantly in the 3rd week of IH exposures. These effects were significantly exacerbated in MT-KO mice, but completely prevented in MT-TG mice. In vitro mechanistic study with H9c2 cardiac and primary neonatal cardiomyocytes showed that MT protection from ER stress-induced apoptosis was mediated by up-regulating Akt phosphorylation since inhibition of Akt phosphorylation abolished MT's protection MT from ER stress and apoptosis. These findings suggest that chronic IH is able to induce cardiac ER stress, cell death and inflammation can be prevented by MT, probably via up-regulation of Akt function.

Topics: Animals; Apoptosis; Cell Death; Cell Line; Drug Administration Schedule; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Gene Deletion; Gene Expression Regulation; Hypoxia; Male; Metallothionein; Mice; Myocardium; Oxygen; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; Stress, Physiological

To study the relationship between hypoxia and metallothionein expression in bladder biopsies of interstitial cystitis/painful bladder syndrome patients.. The study group consisted of 41 patients with interstitial cystitis/painful bladder syndrome, and the control group consisted of 12 volunteers without any interstitial cystitis/painful bladder syndrome symptoms. All biopsy specimens were analyzed for both proteins of hypoxia-inducible factor-1α and metallothionein expression by immunoblotting, immunostaining and confocal laser scanning microscopy. Data were analyzed using the Mann-Whitney U-test.. An increased expression of hypoxia-inducible factor-1α and metallothionein was noted in the study group compared with the control group (P < 0.05). Both proteins of hypoxia-inducible factor-1α and metallothionein mainly distributed over bladder urothelium by immunohistochemical staining, and showed co-localization under confocal microscopy.. High expression and co-localization of metallothionein and hypoxia-inducible factor-1 alpha in the bladder mucosa of patients with interstitial cystitis suggest that overexpression of metallothionein is associated with the bladder hypoxia related to this disease.

Topics: Adult; Biopsy; Cystitis, Interstitial; Female; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Metallothionein; Middle Aged; Urinary Bladder; Urothelium

Hypoxic damage complicates islet isolation for transplantation and may contribute to beta cell failure in type 2 diabetes. Polymorphisms in the SLC30A8 gene, encoding the secretory granule zinc transporter 8 (ZnT8), influence type 2 diabetes risk, conceivably by modulating cytosolic Zn(2+) levels. We have therefore explored the role of ZnT8 and cytosolic Zn(2+) in the response to hypoxia of pancreatic islet cells.. Human, mouse or rat islets were isolated and exposed to varying O2 tensions. Cytosolic free zinc was measured using the adenovirally expressed recombinant targeted zinc probe eCALWY4. Gene expression was measured using quantitative (q)RT-PCR, western (immuno-) blotting or immunocytochemistry. Beta cells were identified by insulin immunoreactivity.. Deprivation of O2 (1% vs 5% or 21%) for 24 h lowered free cytosolic Zn(2+) concentrations by ~40% (p < 0.05) and ~30% (p < 0.05) in mouse and human islet cells, respectively. Hypoxia similarly decreased SLC30A8 mRNA expression in islets, and immunoreactivity in beta cells. Implicating lowered ZnT8 levels in the hypoxia-induced fall in cytosolic Zn(2+), genetic ablation of Slc30a8 from mouse islets lowered cytosolic Zn(2+) by ~40% (p < 0.05) and decreased the induction of metallothionein (Mt1, Mt2) genes. Cell survival in the face of hypoxia was enhanced in small islets of older (>12 weeks) Slc30a8 null mice vs controls, but not younger animals.. The response of pancreatic beta cells to hypoxia is characterised by decreased SLC30A8 expression and lowered cytosolic Zn(2+) concentrations. The dependence on ZnT8 of hypoxia-induced changes in cell survival may contribute to the actions of SLC30A8 variants on diabetes risk in humans.

Topics: Animals; Cation Transport Proteins; Cytosol; Humans; Hypoxia; Insulin-Secreting Cells; Islets of Langerhans; Metallothionein; Mice; Rats; Zinc; Zinc Transporter 8

The present study was to explore the effect of metallothionein (MT) on intermittent hypoxia (IH) induced aortic pathogenic changes. Markers of oxidative damages, inflammation, and vascular remodeling were observed by immunohistochemical staining after 3 days and 1, 3, and 8 weeks after IH exposures. Endogenous MT was induced after 3 days of IH but was significantly decreased after 8 weeks of IH. Compared with the wild-type mice, MT knock-out mice exhibited earlier and more severe pathogenic changes of oxidative damages, inflammatory responses, and cellular apoptosis, as indicated by the significant accumulation of collagen, increased levels of connective tissue growth factor, transforming growth factor β1, tumor necrosis factor-alpha, vascular cell adhesion molecule 1,3-nitrotyrosine, and 4-hydroxy-2-nonenal in the aorta. These findings suggested that chronic IH may lead to aortic damages characterized by oxidative stress and inflammation, and MT may play a pivotal role in the above pathogenesis process.

Topics: Aldehydes; Animals; Aorta; Apoptosis; Connective Tissue Growth Factor; Hypoxia; Metallothionein; Mice; Mice, Knockout; NADPH Oxidases; Nitric Oxide Synthase Type III; Oxidative Stress; Time Factors; Tumor Necrosis Factor-alpha; Tyrosine

We examined the effect of subchronic hypobaric hypoxia in rat heart. Adult male Sprague-Dawley rats were exposed at 25,000 ft for different time periods (2 and 5 days). Susceptibility of their hearts to oxidative stress as well as modulation in gene expression was evaluated. The results showed a crosstalk between reactive oxygen species (ROS) and nitric oxide (NO), initial response was accompanied by increase in ROS generation and development of oxidative stress as confirmed by increased lipid peroxidation, protein oxidation and accumulation of 2, 4-dinitrophenyl hydrazine and 4-hydroxy-2-nonenal adducts. At the same time, glutathione activity decreased; however, antioxidant enzymatic activities of superoxide dismutases, glutathione-S-transferase, and glutathione peroxidase rose in response to 5-days hypoxia. Interestingly, NO level increased till 5 days, however ROS decreased after 5 days; this observation suggests that ROS/NO balance plays an important role in cardioprotection. This observation is further supported by upregulation of antioxidant genes hemeoxygenase (HO-1) and metallothionein (MT). In addition, hypoxia also induces gradual upregulation of hypoxia-inducible transcription factor (HIF-1α), which in turn induces the expression of adaptive genes erythropoiesis, vascular endothelial growth factor, glucose transporter-1, nitric oxide synthase. Collectively, our data suggests a reciprocal regulation of ROS and NO and this effect is mediated by the increase in antioxidant proteins HO-1 and MT. Along with this HIF-1-mediated induction of various cardioprotective genes also plays an important role in acclimatization.

Topics: Animals; Antioxidants; Heart; Hypoxia; Male; Metallothionein; Myocardium; Oxidative Stress; Rats; Rats, Sprague-Dawley

Protein transduction domains (PTDs), also known as cell-penetrating peptides (CPPs), have been developed as effective systems for delivering bio-active cargos such as proteins, genes and particles. Further improvements on cell-specific targeting, intracellular organelle targeting and intracellular retention are still necessary to enhance the therapeutic effect of PTD fusion proteins. In order to enhance the cell transduction and retention of anti-oxidative metallothionein protein (MT), MT was recombinantly fused with transcriptional activator (Tat) with or without a short peptide (sMTS) derived from mitochondria malate dehydrogenase (mMDH). Cellular uptake and retention time of fusion protein were significantly increased in the H9c2 cell by sMTS. The Tat-sMTS-MT (TMM) fusion protein protected H9c2 cells more effectively against hypoxia, hyperglycemia and combination compared with Tat-MT (TM) by reducing intracellular ROS level. It maintained the normal blood glucose level over an extended period of time in a streptozotocin-induced diabetic mouse model. PTD-sMTS-MT fusion protein has a potential to be used as a therapeutic protein for the treatment or prevention of diabetes and diabetic complications.

Topics: Animals; Cell Line; Cell-Penetrating Peptides; Diabetes Mellitus, Experimental; Gene Products, tat; Hyperglycemia; Hypoglycemic Agents; Hypoxia; Malate Dehydrogenase; Metallothionein; Mice; Mice, Inbred BALB C; Myocytes, Cardiac; Oligopeptides; Rats; Reactive Oxygen Species; Recombinant Fusion Proteins; Transduction, Genetic

The molecular basis of failure to progress in labor is poorly understood. This study was undertaken to characterize the myometrial transcriptome of patients with an arrest of dilatation (AODIL).. Human myometrium was prospectively collected from women in the following groups: (1) spontaneous term labor (TL; n=29) and (2) arrest of dilatation (AODIL; n=14). Gene expression was characterized using Illumina® HumanHT-12 microarrays. A moderated Student's t-test and false discovery rate adjustment were used for analysis. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) of selected genes was performed in an independent sample set. Pathway analysis was performed on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database using Pathway Analysis with Down-weighting of Overlapping Genes (PADOG). The MetaCore knowledge base was also searched for pathway analysis.. (1) Forty-two differentially expressed genes were identified in women with an AODIL; (2) gene ontology analysis indicated enrichment of biological processes, which included regulation of angiogenesis, response to hypoxia, inflammatory response, and chemokine-mediated signaling pathway. Enriched molecular functions included transcription repressor activity, heat shock protein (Hsp) 90 binding, and nitric oxide synthase (NOS) activity; (3) MetaCore analysis identified immune response chemokine (C-C motif) ligand 2 (CCL2) signaling, muscle contraction regulation of endothelial nitric oxide synthase (eNOS) activity in endothelial cells, and triiodothyronine and thyroxine signaling as significantly overrepresented (false discovery rate <0.05); (4) qRT-PCR confirmed the overexpression of Nitric oxide synthase 3 (NOS3); hypoxic ischemic factor 1A (HIF1A); Chemokine (C-C motif) ligand 2 (CCL2); angiopoietin-like 4 (ANGPTL4); ADAM metallopeptidase with thrombospondin type 1, motif 9 (ADAMTS9); G protein-coupled receptor 4 (GPR4); metallothionein 1A (MT1A); MT2A; and selectin E (SELE) in an AODIL.. The myometrium of women with AODIL has a stereotypic transcriptome profile. This disorder has been associated with a pattern of gene expression involved in muscle contraction, an inflammatory response, and hypoxia. This is the first comprehensive and unbiased examination of the molecular basis of an AODIL.

Topics: Angiopoietin-Like Protein 4; Angiopoietins; Chemokine CCL2; Female; Gene Expression; Gene Expression Profiling; Humans; Hypoxia; Inflammation; Labor Stage, First; Metallothionein; Muscle Contraction; Myometrium; Nitric Oxide Synthase; Obstetric Labor Complications; Pregnancy; Prospective Studies

Subcutaneous site is ideal for clinical islet transplantation because it has the advantage of simple operation procedure under local anesthesia and can be biopsied when needed. However, the transplantation outcomes at subcutaneous site have been disappointing due to hypoxia-induced oxidative stress by poor vascularization. We hypothesized that subcutaneously transplanted islets would have hypoxia resistance by using internalization of metallothionein (MT), an antioxidant scavenging enzyme, which was mediated by fusion between MT and cell penetrating Tat peptide. The Tat-MT was dose-dependently transduced into islets without any damage. Tat-MT-treated islets could be protected from oxidative stress induced by intracellular nitric oxide donor, sodium nitroprusside (SNP). When Tat-MT-treated islets were subcutaneously transplanted into diabetic nude mice, they normally controlled the blood glucose levels without severe fluctuation (median survival time (MST): >30 days), whereas most untreated islets were rejected (MST 17 days). From the intraperitoneal glucose tolerance test 5 days after posttransplantation, glucose responsiveness of Tat-MT-treated islets was similar to that of normal healthy mice, while untreated islets had delayed glucose responsiveness. From the results of immunohistochemical stain, Tat-MT-treated islets had strong anti-insulin positive cells and lower anti-HIF-1α positive cells. However, untreated islets had rare anti-insulin positive cells and strong anti-HIF-1α-positive cells. Collectively, these findings demonstrated that Tat-MT delivery into islet could offer a new strategy for successful islet transplantation under subcutaneous space.

Topics: Amino Acid Sequence; Animals; Antioxidants; Gene Products, tat; Glucose; Glucose Tolerance Test; Humans; Hypoxia; Insulin; Islets of Langerhans; Islets of Langerhans Transplantation; Male; Metallothionein; Mice; Molecular Sequence Data; Oxidative Stress; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins

Exposure to high altitude results in hypobaric hypoxia which is considered as an acute physiological stress and often leads to high altitude maladies such as high altitude pulmonary edema (HAPE) and high altitude cerebral edema (HACE). The best way to prevent high altitude injuries is hypoxic preconditioning which has potential clinical usefulness and can be mimicked by cobalt chloride. Preconditioning with cobalt has been reported to provide protection in various tissues against ischemic injury. However, the effect of preconditioning with cobalt against high altitude induced pulmonary edema has not been investigated in vivo. Therefore, in the present study, rats pretreated with saline or cobalt (12.5mg/kg body weight) for 7days were exposed to hypobaric hypoxia of 9142m for 5h at 24°C. Formation of pulmonary edema was assessed by measuring transvascular leakage of sodium fluorescein dye and lung water content. Total protein content, albumin content, vascular endothelial growth factor (VEGF) and cytokine levels were measured in bronchoalveolar lavage fluid. Expression of HO-1, MT, NF-κB DNA binding activity and lung tissue pathology were evaluated to determine the effect of preconditioning on HAPE. Hypobaric hypoxia induced increase in transvascular leakage of sodium fluorescein dye, lung water content, lavage total protein, albumin, VEGF levels, pro-inflammatory cytokine levels, tissue expression of cell adhesion molecules and NF-κB DNA binding activity were reduced significantly after hypoxic preconditioning with cobalt. Expression of anti-inflammatory protein HO-1, MT, TGF-β and IL-6 were increased after hypoxic preconditioning. These data suggest that hypoxic preconditioning with cobalt has protective effect against HAPE.

Topics: Albumins; Altitude; Animals; Bronchoalveolar Lavage Fluid; Capillary Permeability; Cell Adhesion Molecules; Cobalt; Cytokines; DNA; Gene Expression Regulation, Enzymologic; Heme Oxygenase-1; Hypoxia; Lung; Male; Metallothionein; Pulmonary Edema; Rats; Rats, Sprague-Dawley; Transcription Factor RelA; Vascular Endothelial Growth Factor A

Acute systemic hypoxia induces delayed cardioprotection against ischaemia-reperfusion injury in the heart. As cobalt chloride (CoCl₂) is known to elicit hypoxia-like responses, it was hypothesized that this chemical would mimic the preconditioning effect and facilitate acclimatization to hypobaric hypoxia in rat heart.. Male Sprague-Dawley rats treated with distilled water or cobalt chloride (12.5 mg Co/kg for 7 days) were exposed to simulated altitude at 7622 m for different time periods (1, 2, 3 and 5 days).. Hypoxic preconditioning with cobalt appreciably attenuated hypobaric hypoxia-induced oxidative damage as observed by a decrease in free radical (reactive oxygen species) generation, oxidation of lipids and proteins. Interestingly, the observed effect was due to increased expression of the antioxidant proteins hemeoxygenase and metallothionein, as no significant change was observed in antioxidant enzyme activity. Hypoxic preconditioning with cobalt increased hypoxia-inducible factor 1α (HIF-1α) expression as well as HIF-1 DNA binding activity, which further resulted in increased expression of HIF-1 regulated genes such as erythropoietin, vascular endothelial growth factor and glucose transporter. A significant decrease was observed in lactate dehydrogenase activity and lactate levels in the heart of preconditioned animals compared with non-preconditioned animals exposed to hypoxia.. The results showed that hypoxic preconditioning with cobalt induces acclimatization by up-regulation of hemeoxygenase 1 and metallothionein 1 via HIF-1 stabilization.

Topics: Acclimatization; Animals; Cardiotonic Agents; Cobalt; DNA-Binding Proteins; Erythropoietin; Heart; Heme Oxygenase-1; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Metallothionein; Myocardium; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury; Up-Regulation; Vascular Endothelial Growth Factor A

Shukla, Dhananjay, Saurabh Saxena, Purushotman Jayamurthy, Mustoori Sairam, Mrinalini, Singh, Swatantra Kumar Jain, Anju Bansal, and Govindaswamy Ilavazaghan. High Alt. Med. Biol. 10:57-69, 2009.-Hypoxic preco759nditioning (HPC) provides robust protection against injury from subsequent prolonged hypobaric hypoxia, which is a characteristic of high altitude and is known to induce oxidative injury in lung by increasing the generation of reactive oxygen species (ROS) and decreasing the effectiveness of the antioxidant defense system. We hypothesize that HPC with cobalt might protect the lung from subsequent hypobaric hypoxia-induced lung injury. HPC with cobalt can be achieved by oral feeding of CoCl(2) (12.5 mg kg(-1)) in rats for 7 days. Nonpreconditioned rats responded to hypobaric hypoxia (7619 m) by increased reactive oxygen species (ROS) generation and a decreased GSH/GSSG ratio. They also showed a marked increase in lipid peroxidation, heat-shock proteins (HSP32, HSP70), metallothionins (MT), levels of inflammatory cytokines (TNF-alpha, IFN-gamma, MCP-1), and SOD, GPx, and GST enzyme activity. In contrast, rats preconditioned with cobalt were far less impaired by severe hypobaric hypoxia, as observed by decreased ROS generation, lipid peroxidation, and inflammatory cytokine release and an inceased GSH/GSSG ratio. Increased expression of antioxidative proeins Nrf-1, HSP-32, and MT was also observed in cobalt- preconditioned animals. A marked increase in the protein expression and DNA binding activity of hypoxia-inducible transcriptional factor (HIF-1alpha) and its regulated genes, such as erythropoietin (EPO) and glucose transporter-1 (glut-1), was observed after HPC with cobalt. We conclude that HPC with cobalt enhances antioxidant status in the lung and protects from subsequent hypobaric hypoxia-induced oxidative stress.

Topics: Animals; Antimutagenic Agents; Cobalt; Cytokines; Glutathione; Glutathione Disulfide; Heme Oxygenase-1; HSP70 Heat-Shock Proteins; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Ischemic Preconditioning; Lipid Peroxidation; Lung; Lung Injury; Male; Metallothionein; NF-E2-Related Factor 1; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; RNA, Messenger; Superoxide Dismutase; Vascular Endothelial Growth Factor A

Topics: Acute Disease; Animals; Calcium Signaling; Cyclic GMP; Endothelial Cells; Humans; Hypoxia; Metallothionein; Mice; Mice, Knockout; Models, Biological; Nitric Oxide; Nitrosation; Protein Kinase C; Protein Processing, Post-Translational; Pulmonary Artery; Rats; Signal Transduction; Vascular Resistance; Vasoconstriction; Zinc

The effect of combined-factors (hypoxia+copper) on the biochemical parameters and antioxidant defenses were studied in the neotropical fish Piaractus mesopotamicus. Fish were exposed for 48 h to 0.4 mg Cu(2+) L(-1) (0.4Cu), hypoxia=50 mm Hg (Hpx), and 0.4 mg Cu(2) L(-1)+hypoxia=50 mm Hg (0.4CuHpx). The exposure to 0.4Cu increased the reactive oxygen species (ROS) in the liver, accompanied by increases in superoxide dismutase (SOD) and decreases in catalase (CAT) activity, showing the influence of copper in this protection. The exposure to Hpx decreased the activity of glutathione peroxidase (GSH-Px) and CAT. Exposure to a combined-factor caused an increase in the ROS production followed by an increase in SOD and a decrease in GSH-Px and CAT. At 0.4Cu, fish presented a reduction in CAT, while in Hpx decreases in SOD, CAT and GSH-Px were observed in red muscles. Single-factors were insufficient to cause ROS production. In combined-factors, increased ROS formation and decreased SOD, CAT and GSH-Px were observed. RBC increased in all groups, but only under combined-factors was there an increase in hemoglobin. Copper plasma concentration increased in groups exposed to copper. Na(+)/K(+)-ATPase activity in gills decreased in 0.4Cu and 0.4CuHpx, and increased in Hpx. Metallothionein concentration in gills increased under combined-factors. Combined-factors caused significant disturbances in the antioxidant defenses and biochemical parameters than single-factors.

Topics: Animals; Antioxidants; Catalase; Copper Sulfate; Erythrocyte Count; Fish Proteins; Fishes; Gills; Glutathione Peroxidase; Hypoxia; Liver; Metallothionein; Muscle Fibers, Fast-Twitch; Oxidative Stress; Reactive Oxygen Species; Sodium-Potassium-Exchanging ATPase; Superoxide Dismutase; Water Pollutants, Chemical

The metal binding protein metallothionein (MT) is a target for nitric oxide (NO), causing release of bound zinc that affects myogenic reflex in systemic resistance vessels. Here, we investigate a role for NO-induced zinc release in pulmonary vasoregulation. We show that acute hypoxia causes reversible constriction of intraacinar arteries (<50 microm/L) in isolated perfused mouse lung (IPL). We further demonstrate that isolated pulmonary (but not aortic) endothelial cells constrict in hypoxia. Hypoxia also causes NO-dependent increases in labile zinc in mouse lung endothelial cells and endothelium of IPL. The latter observation is dependent on MT because it is not apparent in IPL of MT(-/-) mice. Data from NO-sensitive fluorescence resonance energy transfer-based reporters support hypoxia-induced NO production in pulmonary endothelium. Furthermore, hypoxic constriction is blunted in IPL of MT(-/-) mice and in wild-type mice, or rats, treated with the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), suggesting a role for chelatable zinc in modulating HPV. Finally, the NO donor DETAnonoate causes further vasoconstriction in hypoxic IPL in which NO vasodilatory pathways are inhibited. Collectively, these data suggest that zinc thiolate signaling is a component of the effects of acute hypoxia-mediated NO biosynthesis and that this pathway may contribute to constriction in the pulmonary vasculature.

Topics: Animals; Aorta; Cell Size; Cells, Cultured; Chelating Agents; Endothelial Cells; Ethylenediamines; Hypoxia; In Vitro Techniques; Metallothionein; Mice; Mice, Transgenic; Microscopy, Fluorescence; Nitric Oxide; Nitrosation; Organ Specificity; Oxygen; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Sheep; Vascular Resistance; Vasoconstriction; Zinc

Tumor hypoxia is a common feature of several cancers, including prostate cancer, and is associated with tumor progression, acquisition of anti-apoptotic potential and therapeutic resistance. We explored hypoxia-inducible genes and examined the effect of knockdown of a target molecule with small interference RNA (siRNA) on the proliferation of human prostate cancer cells. Human prostate cancer cell lines (LNCaP and PC-3) were cultured in normoxia (21% O2) or hypoxia (0.5% O2). Hypoxia-inducible genes were identified by cDNA microarray analysis. Metallothionein (MT) expression was assessed by real-time RT-PCR, Western blot analysis and immunohistochemical staining. siRNA was transfected to knock down MT expression, and the cell cycle and apoptosis were evaluated by flow cytometry analysis. In cDNA microarray analysis, 22 genes (including MT) were up-regulated under hypoxia. MT-1X and MT-2A were up-regulated in real-time RT-PCR. In particular, MT-2A was increased 3-fold in LNCaP and 8-fold in PC-3. The siRNA-MT-2A treatment resulted in a 20% inhibition of cell growth and induced apoptosis in both LNCaP and PC-3. In human prostate tissue, intense staining of MT was observed in cancer cells and residual cancer cells after androgen ablation therapy, while normal tissue was only stained in patches. In conclusion, MT was up-regulated under hypoxia in prostate cancer cells and overexpressed in prostate cancer tissue and residual cancer cells after androgen ablation therapy. As down-regulation of MT by siRNA inhibited cell growth and induced cell death, MT may be a new molecular target for the treatment of human prostate cancer.

Topics: Aged; Androgen Antagonists; Androgens; Apoptosis; Biomarkers, Tumor; Blotting, Western; Cell Survival; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Hypoxia; Male; Metallothionein; Middle Aged; Oligonucleotide Array Sequence Analysis; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Transfection; Tumor Cells, Cultured; Up-Regulation

To develop a potent hypoxia-inducible promoter, we evaluated the usefulness of chimeric combinations of the (Egr-1)-binding site (EBS) from the Egr-1 gene, the metal-response element (MRE) from the metallothionein gene, and the hypoxia-response element (HRE) from the phosphoglycerate kinase 1 gene. In transient transfection assays, combining three copies of HRE (3 x HRE) with either EBS or MRE significantly increased hypoxia responsiveness. When a three-enhancer combination was tested, the EBS-MRE-3 x HRE (E-M-H) gave a hypoxia induction ratio of 69. The expression induced from E-M-H-pGL3 was 2.4-fold higher than that induced from H-pGL3 and even surpassed the expression from a human cytomegalovirus promoter-driven vector. The high inducibility of E-M-H was confirmed by validation studies in different cells and by expressing other cDNAs. Gel shift assays together with functional overexpression studies suggested that increased levels of hypoxia-inducible factor 1alpha, metal transcription factor-1 and Egr-1 may be associated with the high inducibility of the E-M-H chimeric promoter. E-M-H was also induced by hypoxia mimetics such as Co2+ and deferoxamine (DFX) and by hydrogen peroxide. Gene expression from the E-M-H was reversible as shown by the reduced expression of the transgene upon removal of inducers such as hypoxia and DFX. In vivo evaluation of the E-M-H in ischemic muscle revealed that erythropoietin secretion and luciferase and LacZ expression were significantly higher in the E-M-H group than in a control or H group. With its high induction capacity and versatile means of modulation, this novel chimeric promoter should find wide application in the treatment of ischemic diseases and cancer.

Topics: Animals; Cell Line; Cell Line, Tumor; Chimera; DNA-Binding Proteins; DNA, Single-Stranded; Electrophoretic Mobility Shift Assay; Enzyme-Linked Immunosorbent Assay; Genetic Engineering; HeLa Cells; Hindlimb; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Laser-Doppler Flowmetry; Metallothionein; Metals; Mice; Phosphoglycerate Kinase; Promoter Regions, Genetic; Regional Blood Flow; Transcription Factor MTF-1; Transcription Factors; Transfection

The molecular response to hypoxia stress in aquatic invertebrates remains relatively unknown. In this study, we investigated the response of the Pacific oyster Crassostrea gigas to hypoxia under experimental conditions and focused on the analysis of the differential expression patterns of specific genes associated with hypoxia response. A suppression subtractive hybridization method was used to identify specific hypoxia up- and downregulated genes, in gills, mantle and digestive gland, after 7-10 days and 24 days of exposure. This method revealed 616 different sequences corresponding to 12 major physiological functions. The expression of eight potentially regulated genes was analysed by RT-PCR in different tissues at different sampling times over the time course of hypoxia. These genes are implicated in different physiological pathways such as respiration (carbonic anhydrase), carbohydrate metabolism (glycogen phosphorylase), lipid metabolism (delta-9 desaturase), oxidative metabolism and the immune system (glutathione peroxidase), protein regulation (BTF3, transcription factor), nucleic acid regulation (myc homologue), metal sequestration (putative metallothionein) and stress response (heat shock protein 70). Stress proteins (metallothioneins and heat shock proteins) were also quantified. This study contributes to the characterization of many potential genetic markers that could be used in future environmental monitoring, and could lead to explore new mechanisms of stress tolerance in marine mollusc species.

Topics: Animals; Gene Expression Regulation; HSP70 Heat-Shock Proteins; Hypoxia; Metallothionein; Ostreidae; Pacific Ocean

Pimonidazole binding (hypoxia) and involucrin expression (differentiation) overlap extensively in squamous cell carcinomas. This study asks whether involucrin might serve as an endogenous marker for tumor hypoxia. A second question is whether differentiation affects hypoxia-inducible metallothionein (MT) expression in normal human epithelia and squamous cell carcinomas as it does in rodent epithelia.. Thirty-four patients with squamous cell carcinoma of the uterine cervix were infused with pimonidazole hydrochloride solution. The next day, multiple biopsies were formalin-fixed, paraffin-embedded and sectioned at 4 micro m. Qualitative and quantitative analyses for involucrin expression, pimonidazole binding, and human MT-IIa mRNA expression were performed.. No overall correlation between the extent of involucrin expression and pimonidazole binding was observed. The lack of correlation was because of heterogeneous patterns of immunostaining for involucrin generally related to tumor grade. Colocalized immunostaining for involucrin and pimonidazole binding was observed in intermediate grade tumors but not in well-differentiated or poorly differentiated tumors. Human MT-IIa mRNA and MT protein were expressed in basal lamina of normal human epithelia and in the proliferative rims of tumor nests.. Colocalization of immunostaining for involucrin and pimonidazole binding is consistent with oxygen regulation, but the lack of involucrin expression in hypoxic regions of poorly differentiated tumors indicates that its transcriptional status with respect to hypoxia induction is altered by cell differentiation. The localization of MT message and protein in the outer rims of most tumor nests indicates that the transcriptional status of metallothionein is also altered by differentiation.

Topics: Biopsy; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line, Tumor; Female; Humans; Hypoxia; Immunohistochemistry; In Situ Hybridization; Metallothionein; Nitroimidazoles; Oxygen; Prognosis; Protein Precursors; Radiation-Sensitizing Agents; RNA, Messenger; Transcription, Genetic; Uterine Cervical Neoplasms

To study possible mechanisms for metallothionein (MT) inhibition of ischemia-reperfusion-induced myocardial injury, cardiomyocytes isolated from MT-overexpressing transgenic neonatal mouse hearts and nontransgenic controls were subjected to 4 h of hypoxia (5% CO2-95% N2, glucose-free modified Tyrode's solution) followed by 1 h of reoxygenation in MEM + 20% fetal bovine serum (FBS) (5% CO2-95% air), and cytochrome c-mediated caspase-3 activation apoptotic pathway was determined. Hypoxia/reoxygenation-induced apoptosis was significantly suppressed in MT-overexpressing cardiomyocytes, as measured by both terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling and annexin V-FITC binding. In association with apoptosis, mitochondrial cytochrome c release, as determined by Western blot, was observed to occur in nontransgenic cardiomyocytes. Correspondingly, caspase-3 was activated as determined by laser confocal microscopic examination with the use of FITC-conjugated antibody against active caspase-3 and by enzymatic assay. The activation of this apoptotic pathway was significantly inhibited in MT-overexpressing cells, as evidenced by both suppression of cytochrome c release and inhibition of caspase-3 activation. The results demonstrate that MT suppresses hypoxia/reoxygenation-induced cardiomyocyte apoptosis through, at least in part, inhibition of cytochrome c-mediated caspase-3 activation.

Topics: Animals; Apoptosis; Caspase 3; Caspases; Cells, Cultured; Cytochrome c Group; Gene Expression; Hypoxia; In Situ Nick-End Labeling; L-Lactate Dehydrogenase; Metallothionein; Mice; Mice, Transgenic; Mitochondria; Muscle Fibers, Skeletal; Myocardial Reperfusion Injury; Myocardium

Global hypoxia preconditioning provides neuroprotection against a subsequent, normally damaging challenge. While the mechanistic pathways are unknown, changes in the expression of stress-related proteins are implicated. Hypoxia preconditioning attenuates the brain edema and neuropathology associated with kainic acid-induced status epilepticus in a protein synthesis-dependent manner when a kainic acid challenge is given up to one week post-preconditioning. Kainic acid initiates a glutamate-driven status epilepticus causing a Ca2+ and oxidative stress, resulting in injury to the piriform cortex and hippocampus. Stress-related gene expression [e.g. metallothioneins (MTs), heme oxygenase-1 (HO-1)] is enhanced during seizures in vulnerable brain areas, (e.g. piriform cortex). This study explores the effects of hypoxia preconditioning on expression of MT-1, MT-2 and HO-1 before and after kainic acid-induced seizures. Analysis of MT-1, MT-2 and HO-1 expression, through Western and Northern blotting, indicates that there is a variable pattern of induction and suppression of these two genes following hypoxia preconditioning alone as well as after kainic acid-induced seizures compared to non-preconditioned animals. These findings suggest that hypoxia preconditioning induces an adaptive response that prevents kainic acid seizure-associated neuropathology even when robust seizures occur. This may involve a variety of stress-related proteins, working in concert, each with their own individual expression profiles. Induction of this type of neuroprotection pharmacologically, or through preconditioning, will provide a better understanding of the stress response in brain.

Topics: Animals; Cerebral Cortex; Disease Models, Animal; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Hippocampus; Hypoxia; Kainic Acid; Male; Metallothionein; Rats; Rats, Wistar; Seizures; Time Factors

In order to evaluate whether tumor microenvironment might influence the response of the metallothionein promoter to heavy-metal exposure, we transfected HT-29 colon carcinoma cells with the vector phMTIIA-CAT-neo, containing a fusion gene consisting of 426 bp of the human metallothionein-IIa (hMT-IIA) promoter immediately upstream of the bacterial chloramphenicol acetyl transferase (CAT) gene. We grew one of the stable transfectants (clone 20) as three-dimensional multicell tumor spheroids, exposed them to CdCl2 and measured CAT expression in cells isolated from various depths into the spheroids. Cellular populations were isolated by flow cytometry on the basis of Hoescht 33342 fluorescence intensity, taking advantage of the dye's diffusion gradient to isolate cells from inner (dim) and outer (bright) regions of stained spheroids. When intact spheroids were incubated for 18 h in the presence of 5 microM CdCl2, CAT activity was induced in all cell fractions isolated from the spheroids, but induction was 10-fold greater in cells in the outermost fraction (fraction 10) than inner fraction (fraction 2). When spheroids were dissociated, sorted into individual fractions and then incubated with cadmium, CAT expression was maximized in all fractions. Exposure of intact spheroids to 30 microM CdCl2 resulted in increased CAT induction in cells isolated from the internal fractions of the spheroids. The data suggest that limited diffusion of cadmium through cells organized in a tissue-like arrangement may account for the lower levels of hMT-IIA promoter activity observed in cells collected from increasing depths into the spheroids.

Topics: Cadmium; Chloramphenicol O-Acetyltransferase; Gene Expression Regulation; Humans; Hydrogen-Ion Concentration; Hypoxia; In Vitro Techniques; Metallothionein; Organoids; Promoter Regions, Genetic; Transfection; Tumor Cells, Cultured